Bro1 directly stimulates Vps4 activity to promote Intralumenal Vesicle Formation during Multivesicular Body biogenesis

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) form intralumenal vesicles (ILVs) during the conversion of endosomes to multivesicular bodies (MVBs). The assembly and disassembly of an ESCRT-III polymer facilitates membrane remodeling and scission during this process. The ESCRT-III-associated protein Bro1 (the yeast homolog of mammalian proteins ALIX and HD-PTP) promotes ESCRT-III assembly and inhibits disassembly of ESCRT-III filaments by impeding Vps4, a AAA-ATPase that dismantles ESCRT-III polymers. Here we show that the evolutionarily conserved "V domain" of Bro1- family proteins directly stimulate Vps4 ATPase activity and this activity is enhanced by interaction with ubiquitin. Surprisingly, a carboxyl-terminal fragment of Bro1 containing the V domain supports ILV formation but not sorting of cargo into ILVs, revealing that these two processes can be uncoupled. These studies implicate Bro1 as a factor coordinating cargo sorting with direct regulation of Vps4 to modulate ESCRT-III driven ILV formation during MVB biogenesis.